Sporotrichosis: In silico design of new molecular markers for the Sporothrix genus

ABSTRACT Background: Sporotrichosis, a cosmopolitan mycosis caused by dimorphic fungi of the Sporothrix complex, affects humans and animals. This study aimed to develop new molecular markers for Sporothrix genome detection in biological samples using PCR. Methods: A specific region of DNA sequences from the Sporothrix genus, publicly available in GenBank, was chosen for primer design. After testing the in silico specificity of these primers, in vitro specificity was evaluated using the PCR technique. Results: Three specific primers with 100% specificity for the Sporothrix genus were generated. Conclusions: PCR using the designed primers can be used to develop molecular diagnostics for sporotrichosis.

Sporotrichosis is a cosmopolitan subcutaneous mycosis that affects humans and animals and is common in tropical and subtropical regions 1,2 . The disease is caused by thermally dimorphic fungal complexes of Sporothrix, which comprises S. brasiliensis, S. globosa, S. schenckii, S. luriei, and S. mexicana species. Other known species of this genus that share genetic similarities include S. pallida, S. nivea, and S. albican 3 . The species S. brasiliensis is the most virulent of this genus and is commonly related to zoonotic transmission, whereas S. schenckii and S. globosa are mostly isolated based on environmental transmission 3,4 .
Mycosis diagnoses are time-consuming. Isolation and visualization of the agent in biological samples are considered the gold standard, but this is challenging 5,6 . The preferred agar for fungal culture is Sabouraud dextrose owing to its low cost and its widespread ability to yield growth of most fungi 6 . However, growth can be slow and the development of characteristic structures used to identify these fungi is non-specific. Culture growth can take up to 10 days depending on the clinical sample; isolation and identification can take twice that amount of time. Various serologic methods can be used to assist diagnosis by identifying atypical forms of sporotrichosis or as a method of healing control. Some serologic tests used in this context include the highly specific and sensitive latex agglutination, immunodiffusion, the complement fixation test, ELISA, western blotting, and immunofluorescence 5 .
Biomolecular technology and bioinformatics have become powerful tools facilitating the creation of specific biomarkers for each pathogen 7 . In this context, polymerase chain reaction (PCR) has become a powerful tool to speed up the diagnosis process. Owing to the sensitivity afforded by PCR, diagnosis is possible in the earliest stages of infection, and this was shown to be a reliable strategy for more efficient and refined diagnostics 7,8 . Kano et al. (2005) reported Sporothrix DNA detection and sex-level identification using skin biopsies from cats with sporotrichosis 8 . Successful detection and identification of Sporothrix spp. via PCR in cats are relevant because felines are essential transmission vectors for humans. Detection using PCR is thus helpful for the prevention of these infections.
PCR-based assays are fast, easy to perform, simple to interpret, and highly discriminatory 7 . Nested PCR, for example, is an enhanced PCR reaction that allows for the amplification of products that are otherwise not visualized in a single PCR reaction 9 . In this technique the amplified fragment in the first reaction called "Out" is further amplified to allow fragments to be finally visualized in the second reaction called "In." Nested PCR consists of two steps, wherein approximately 1 μL of the amplified PCR product from the "Out" step is used for the second reaction "In." This technique showed a sensitivity of 40 fg of S. schenckii DNA, detecting up to only one colony-forming unit in tissue samples 7,9 . Isolates from different parts of the world were tested, and the nested PCR showed high sensitivity and specificity. A study by Hu et al., 2003, indicated that nested PCR is 100 times more sensitive than the first PCR, suggesting that it is a highly sensitive and specific method for detecting S. schenckii target DNA in biological samples 10 .
The development of PCR for the diagnosis of sporotrichosis is vital because the gold-standard diagnosis is time-consuming and not entirely reliable. Accordingly, this in silico study presents new molecular markers that anneal to the Internal Transcribed sequences (ITS) of the ribosomal DNA (rDNA) domains specific to the Sporothrix genus, which can be used to support species identification and disease diagnostics.
An rDNA sequence from genus Sporothrix, deposited in GenBank, a genomic non-redundant database with available data, was selected from https://www.ncbi.nlm.nih.gov/nuccore/KP132788.1/. The partial sequence of 18S region, complete ITS1 sequence, complete 5.8S sequence, and partial sequence of 28S region were used to design specific primers. In terms of the sequence, the primers were designed using the Primer-BLAST tool 11 . After primer design, a search was performed to evaluate their specificity for the genus Sporothrix, using BLASTn software available at (https://blast.ncbi.nlm. nih.gov). The primers designed were analyzed in silico to verify their complementarity with the genomes of other organisms, especially other pathogenic fungi, such as Aspergillus spp., Histoplasma spp., Candida spp., Coccidioides immitis, Blastomyces dermatitidis, Cryptococcus spp., Paracoccidioides spp., and species of melanogenic fungi of the family Herpothrichiellaceae. Complementarity with the human genome was also verified, as these markers will be used to detect the fungal agent in human biological samples.
After in silico analyses, in vitro tests were performed to confirm the specificity of the designed primers. A validation protocol for the PCR reaction using these primers was not carried out in this study. Thus, only eight biological biospsy samples obtained from patients treated at the Santa Casa Hospital of Belo Horizonte, Minas Gerais, Brazil, were analyzed using the nested-PCR technique. The patients presented clinical and laboratory diagnosis for sporotrichosis. The present study was approved by CONEP through the Research Ethics Committee of Santa Casa de Belo Horizonte Hospital -CAAE: 55549216.2.0000.5138. Total DNA was extracted and purified from the biopsy samples of the patients using the PureLink™ Genomic DNA Mini Kit, Invitrogen kit (USA). Thereafter, the whole DNA was quantified via spectrophotometry using a Nanovue Plus (GE Healthcare Life Sciences, Sweden).
The DNA extracted from the clinical samples was subjected to PCR reaction using the primers designed. The reaction was performed with a final volume of 10 μL in a solution containing PCR 1´ buffer, 3 mM MgCl 2 , 0.2 μM dNTPs, 1.0 U/μL Taq DNA polymerase (all reagents were from Ludwig, Brazil), 0.2 μM of each forward and reverse primer (Invitrogen, USA) and Milli-Q water; 1 μL of extracted DNA (20.0 ng/μL) was used for each reaction. The amplification cycles were as follows: one first step of 95°C for 5 min, followed by 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s; with a final step of 72°C for 5 min.
Negative and positive PCR controls were included in each reaction. The positive control was DNA extracted from an international standard culture of Sporothrix schenkii ATCC MF765977, a reference for all reactions. Mixtures containing all reaction components were used as the negative control, with sterile ultrapure water in place of the extracted DNA. The final amplification product was analyzed on a 7% polyacrylamide gel stained with silver nitrate.
The ITS-18S rDNA was chosen as the genomic region to develop the primers based on a literature search. The "International Society of Human and Animal Mycology (ISHAM) -ITS, reference DNA barcoding database-, the quality standardized tool for routine identification of human and animal pathogenic fungi" was used as the basis to select the coding sequence of the ITS rDNA gene of the genus Sporothrix 12 . BLAST analysis of the primer SPOR2 showed that it aligns only with the Sporothrix genus. This primer was used for both reactions of the nested PCR.
The PCR using primers designed specifically for the Sporothrix genus and the clinical samples, as well as the positive control, yielded bands of approximately 450 bp in the first "Out" PCR and of approximately 250 bp in the second "In" PCR, as predicted in silico. There was no amplification of the negative control (Figure 1). The designed primers showed specificity for the samples tested, namely S. schenkii and S. brasiliensis, with no amplification of bands when DNA samples from other species of fungi were tested (Figure 2).  The design of new primers underlies the innovation of this study, as a new molecular marker is being proposed for the detection of the Sporothrix genome in biological samples. These primers anneal to a specific region of the Sporothrix complex genome, allowing them to be used as an efficient diagnostic marker. The target sequence was chosen because it is a vast region of ITS rDNA (partial 18S, complete ITS1, 5.8S complete, and partial 28S) 12 . Statistical analysis of sequences deposited in GenBank showed that this sequence aligns with sequences from several strains of the Sporothrix genus, including the most common species in Brazil, S. schenckii, S. globosa, and S. brasiliensis, as demonstrated in a study by Zhou et al., 2014 13 . BLASTn analysis showed 100% identity between the target sequence and the sequence from genus Sporothrix, thus confirming that the sequence chosen is reliable for designing specific primers.
BLASTn analysis of the primer SPOR1 showed that it aligns only with the Sporothrix genus and Ophiostoma, considering samples deposited in GenBank to the present day. These species, belonging to the Ophiostomatales order, have been described in previous studies related to Sporothrix 10,14 . Thus, alignment with Ophiostoma sequences does not affect the specificity of the designed primers as Ophiostoma is not of clinical importance. This fungus is isolated from trees with no reports of causing human infection.
BLASTn analysis of the SPOR3 primer demonstrated that it aligns with sequences of several fungal species. Therefore, it should only be used for the "In" reaction of the nested-PCR, so that it will only anneal with the fragment amplified by the first "Out" reaction of nested PCR. Nested PCR is used to amplify products that would not be visualized after only one PCR reaction. Thus, it allows for the visualization of fragments based on more pronounced bands associated with a second reaction, as demonstrated in different studies 8,9,10,12 .
BLASTn analysis of SPOR2 (reverse) primer resulted in 100% specificity. Considering the samples deposited in GenBank until the present time, SPOR2 aligned only with sequences from Sporothrix spp. This primer was used in the "Out" and "In" reactions of the nested PCR. Thus, the in silico analyses demonstrated that these primers are specific and of good quality; hence, they can be used to detect Sporothrix genomes for the diagnosis of sporotrichosis.
Results of the PCR "Out" reaction showed that the tested samples and positive control yielded bands between 400 and 500 base pairs, which corroborates the results from in silico analyses suggesting a fragment of 449 base pairs. The PCR "In" results showed that samples tested, and the positive control yielded bands between 200 and 300 base pairs, which corroborates the results from in silico analyses that suggested a fragment of 260 base pairs. PCR using the designed primers showed 100% specificity, in line with the results from a previous study by Rodriguez et al., 2014 15 . This study indicated that PCR can be a highly specific method for detecting S. schenckii target DNA in biological samples 9 .
These studies indicate that PCR can be a highly sensitive method for detecting Sporothrix target DNA in biological samples, and it can also be used for healing control as it can detect even minimal amounts of DNA in samples. Based on the results showing high specificity among the species compared (100%) and high DNA detection rate, the sequences of the primers SPOR1, SPOR2, and SPOR3 were registered in the National Institute of Property (INPI), under the number BR 10 2017 028202 3. As a final note, this study provides perspectives to continue developing molecular diagnostic methodologies for sporotrichosis and other systemic mycoses as PCR can be applied to a broad spectrum of infectious diseases.